Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals. Natural vanillin which is extracted from orchid Vanilla planifolia beans is relatively expensive. The production of vanilla bean is a lengthy process that is highly dependent on suitable soil and climatic conditions. Beans appear after 4-5 years of cultivation and the aroma is developed in fruit after a long process called “curing” that takes 6 months. The consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. Less than 5% of worldwide vanillin production comes from natural vanilla. Because of the scarcity and expense of natural vanilla extract, there has long been interest in the synthetic preparation of its predominant component. Vanillin (4-hydroxy-3-methoxybenzal-dehyde) is the major organoleptic component of vanilla flavour.
As the demand for vanillin is higher than can be extracted from orchid Vanilla planifolia beans, the remainder is produced by alternative means. Chemical synthesis is the most important source of vanillin. Vanillin was first synthesized from eugenol found in oil of clove and afterward synthesized from lignin containing sulfite liquor, a byproduct of wood pulp processing in paper manufacture. While some vanillin is still made from lignin waste, today most synthetic vanillin is synthesized in a two-step process from the petrochemical precursors: guaiacol and glyoxylic acid. Vanillin can be also produced chemically by molecular breakage of curcumine, eugenol or piperrin.
The large difference between the prices of natural and synthetic vanillin, the increasing customer-led demand for “natural” and “healthy” flavors, and the serving of “natural” marketing claims have been leading to a growing interest of the flavor industry to produce natural vanillin from other natural sources by bioconversion1,2,3,4,5. The use of microbial cells and their enzymes as biocatalysts in the synthesis of fine chemicals has attracted much attention in the field green chemistry and white biotechnology. The products of such bioconversion are considered natural since the European Community Legislation (incorporates products that are produced from biological sources by living cells or their enzymes under the term “natural products”.
Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Although vanillin production via conversion of isoeugenol has been widely reported in various microorganisms, including Aspergillus niger6; strains of the genera Klebsiella, Enterobacter, and Serratia7; Rhodococcus rhodochrous8; Bacillus subtilis B29; Bacillus fusiformis10; B. subtilis HS811; Pseudomonas nitroreducens12; Pseudomonas putida13; Pseudomonas chlororaphis14; Bacillus pumilus15; and Nocardia iowensis16. De novo synthesis from glucose using metabolically engineered yeast strains was recently described17.
S. cerevisiae is a valuable cell factory for production of high-value industrial biotechnological products relies. It is well adapted for bio-refinery processes due to its capacity for cell-recycle fermentation and its remarkable tolerance against various stresses, such as low pH, high temperature, and various inhibitors18. Additionally, S. cerevisiae is an extremely well characterized model organism, facilitating metabolic engineering19,20 due to the availability of the complete genome sequence and detailed characterization of metabolic pathways21.
U.S. Pat. No. 6,372,461B1 describes the synthesis of vanillin from a carbon source, by a microbe-catalyzed conversion step requiring five enzymes which are provided by a recombinant microbe, and an enzyme-catalyzed reduction step to reduce vanillic acid by an aryl-aldehyde dehydrogenase.
EP2388333A2 describes a microbial cell capable of production of vanillin, comprising at least three heterologous enzymatic activities, i.e. 3-dehydroshikimate dehydratase, aromatic carboxylic acid reductase and 3 O-methyl transferase activities.
WO2011124693A1 describes methods of generating gene mosaics by homeologous in vivo recombination, whereby metabolic pathways can be constructed, which do not exist in nature.
US2003/070188 A1 describes a biosynthetic pathway of vanillin that comprises the conversion of p-coumaric acid to p-hydroxybenzaldehyde, and vanillin production in cultured Vanilla planifolia, or transgenic cells and plants having improved vanillin production.
Hansen et al. (Appl Environ Microbiol. 2009; 75(9): 2765-2774) describe de novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharmomyces cerevisiae). The engineered pathways start with dehydroshikimic acid used as a substrate.
Di Gioia et al. (J. Biotechnol. 2011; 156: 309-316) describe metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid.
Brochado et al. (Microbial Cell Factories 2010; 9: 84) describe improved vanillin production in baker's yeast through in silico design.
Priefert et al. (Appl. Microbiol. Biotechnol. 2001; 56: 296-314) describe the biotechnological production of vanillin and the different biosynthesis routes based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids.
Kaur et al. (Appl. Biochem. Microbiol. 2013; 169: 1353-1372) provide a review on biotechnological and molecular approaches for vanillin production.